This invention relates to substantially purified Tumor Necrosis Factor (TNP) Inhibitory Protein and salts, functional derivatives and active fractions thereof, having the ability to inhibit the binding of TNF to its receptors and the cytotoxic effect of TNF and which can be used against the deleterious effects of TNF. It also relates to a process for the purification of said TNF Inhibitory Protein, to its cloning and its production by recombinant DNA techniques. It further relates to pharmaceutical compositions comprising such a protein, or salts, functional derivatives and active fractions thereof; for protecting against the deleterious effects of TNF and to a method of medical treatment using the protein.
Tumor Necrosis Factor-xcex1 (TNF-xcex1) and Lymphotoxin or TNF-xcex2 (hereinafter, TNF refers to both TNF-xcex1 and TNF-xcex2) are cytokines which have many effects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Gresser. Ed.), pp. 83-122, Academic Press, London, and Beutler, B. and Cerami, A. (1987) New England J. Med. 316: 379-385). Both TNF-xcex1 and TNF-xcex2 initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism; they may destroy, for example, tumor cello or virus infected cells and augment antibacterial activities of granulocytes. But, quite clearly, both TNF-xcex1 and TNF-xcex2 have also effects which can be extensively deleterious. There is evidence that over production of TNF-xcex1 can play a major pathogenic role in several diseases. Thus effects of TNF-xcex1, primarily on the vasculature, are now known to be a major cause for symptoms of septic shock (Tracey, K. J. et al. (1986) Science 234: 470-474). In some diseases, TNF may cause excessive lose of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia and TNF-xcex1 was thus called cachectin. It was also described AN a mediator of the damage to tissues in rheumatic diseases (Boutler, op.cit.) and as a major mediator of the damage observed in graft-versus-host reactions.
There is therefore a necessity in finding out ways to eliminate or antagonize endogenously formed or exogenously administered TNT. Our first attempt in this direction was the development of monoclonal antibodies which neutralize the TNF-xcex1 cytotoxic activity and were shown to protect mice against the lethal effect of TNF-xcex1 under conditions mimicking elicitation of septic shock (as described in our U.S. patent application Ser. No. 06/800,262 of Dec. 12, 1985). However, therapy with murine monoclonal antibodies, especially if administered repetitively, may not always be advisable in humans. Therefore the need was felt for development of biological agents which could similarly antagonize the deleterious effects of TNF.
Prior to the filing date of the priority application of the present application, there was no information as to the existence of biological agents which could antagonize the cytotoxic activity of TNF. There were publications describing uromodulin, a 85-kDa immunosuppressive glycoprotein isolated from the urine of pregnant women (Muchmore, Andrew V. and Decker, Jean M. (1985) Science 229:479-481), that was shown to be a high affinity ligand for and a potent Inhibitor of interleukin 1 (IL-1) (Muchmore, Andrew V. and Decker, Jean M. (1986) J. Biol. Chem. 261:13404-13407; Brown, K. M. et al. (1986) Proc. Natl. Acad. Sci. USA 83:9119-9123). Uromodulin was later shown to be identical to the Tamm-Horsfall glycoprotein, the moot abundant protein of renal origin in normal urine (Pennica, Diane et al. (1987) Science 236:83-88). Another inhibitor of IL-1 found in the urine of febrile patients was disclosed in some publications (Liao, Zenghua et al. (1984) J. Exp. Med. 159:126-136; Beckinger, Phillippe et al. (1987) J. Immunol. 139:1546-1549). It was shown that this urine inhibitor of IL-1 affects numerous biological activities of both forms of recombinant IL-1, IL-1xcex1 and IL-1xcex2 to the same extent. Although human TNF-xcex1 shares some of the biological activities of IL-1, this IL-1 Inhibitor did not inhibit the biological activities of TNF-xcex1 (Seckinger, Phillippe et al. (1987) J. Immunol. 139:1541-1545).
Subsequent to the filing date of the priority application of the present application, it was disclosed that uromodulin and the Tamm-Horsfall glycoprotein bind recombinant IL-1xcex1, IL-1xcex2 and TNF-xcex1 In a lectin-like interaction and it was suggested that it may play an important role in the regulation of circulating levels of these lymphokines (Hession, Catherine et al. (1987) Science 237: 1479-1484). Although uromodulin does not inhibit the cytotoxic activity of TNF-xcex1 as monitored by lysis of tumor cell targets, it interacts with recombinant TNF-xcex1 via carbohydrate chains and this interaction may be critical in promoting clearance and/or reducing in vivo toxicity of TNF and other lymphokines (Sherblom, Anne P. (1988) J. Biol. Chem. 263:5418-5424). In a recent publication by Seckinger et al. (J. EXp. Med. (1908) 167:1511-1516) a human inhibitor of TNF-xcex1 obtained from the urine of febrile patients was described as a 40-60 Kda protein inhibiting the cytotoxic activity of TNF-xcex1. It was shown to differ from uromodulin and from the above-mentioned IL-1 inhibitor.
The present invention provides substantially purified TNF Inhibitory Protein which can antagonile the effects of TNF. This antagonism can be determined both by measuring reduction of the cytotoxic activity of TNF as well as by measuring interference with TNF binding to its receptors.
The invention is directed to said TNF Inhibitory Protein in substantially purified form, as well as to its salts, functional derivatives and active fractions thereof.
The invention also relates to a process for the purification of TNF Inhibitory Protein.
The Invention further concerns recombinant DNA molecules comprising the nucleotide sequence coding for said protein, expression vehicles comprising them and host cells transformed therewith.
The substantially purified TNF Inhibitory Protein of the invention in used an the active ingredient of pharmaceutical compositions and in medical treatments to protect mammals against the deleterious affects of TNF.